Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, University of Mustafa Kemal, 31000 Hatay, Turkey.
Cryobiology. 2013 Aug;67(1):70-5. doi: 10.1016/j.cryobiol.2013.05.004. Epub 2013 May 28.
Restoration of male fertility associated with use of the cryopreserved testicular tissue would be a significant advance in human and animal assisted reproductive technology. The purpose of this study was to test the effects of four different cryoprotectant agents (CPA) on spermatogenesis and steroidogenesis in cryopreserved and allotransplanted neonatal mouse testicular tissue. Hank's balanced salt solution (HBSS) with 5% fetal bovine serum including either 0.7 M dimethyl sulfoxide (DMSO), 0.7 M propylene glycol (PrOH), 0.7 M ethylene glycol (EG), or glycerol was used as the cryoprotectant solution. Donor testes were collected and dissected from neonatal pups of CD-1 mice (one day old). Freezing and seeding of the testicular whole tissues was performed using an automated controlled-rate freezer. Four fresh (non-frozen) or frozen-thawed pieces of testes were subcutaneously grafted onto the hind flank of each castrated male NCr nude recipient mouse and harvested after 3 months. Fresh neonatal testes grafts recovered from transplant sites had the most advanced rate of spermatogenesis with elongated spermatid and spermatozoa in 46.6% of seminiferous tubules and had higher levels of serum testosterone compared to all other frozen-thawed-graft groups (p<0.05). Fresh grafts and frozen-thawed grafts in the DMSO group had the highest rate of tissue survival compared to PrOH, EG, and glycerol after harvesting (p>0.05). The most effective CPA for the freezing and thawing of neonatal mouse testes was DMSO in comparison with EG (p<0.05) in both pre-grafted and post-grafted tissues based on histopathological evaluation. Likewise, the highest level of serum testosterone was obtained from the DMSO CPA group compared to all other cryoprotectants evaluated (p<0.05). The typical damage observed in the frozen-thawed grafts included disruption of the interstitial stroma, intercellular connection ruptures, and detachment of spermatogonia from the basement membrane. These findings indicate that neonatal mouse testes were most effectively preserved when frozen with HBSS medium with DMSO and that the type of CPA is a significant factor to obtain the most advanced stages of spermatogenesis and steroidogenesis after cryopreservation, thawing, and transplantation of neonatal mouse testes.
恢复与使用冷冻保存的睾丸组织相关的男性生育能力将是人类和动物辅助生殖技术的重大进展。本研究的目的是测试四种不同的冷冻保护剂（CPA）对冷冻保存和同种异体移植的新生小鼠睾丸组织的生精和类固醇生成的影响。含有 5%胎牛血清的 Hank's 平衡盐溶液（HBSS）中分别含有 0.7 M 二甲亚砜（DMSO）、0.7 M 丙二醇（PrOH）、0.7 M 乙二醇（EG）或甘油，用作冷冻保护剂溶液。供体睾丸从 CD-1 小鼠（1 天大）的新生幼仔中收集并解剖。使用自动化控制速率冷冻机对整个睾丸组织进行冷冻和播种。将 4 个新鲜（未冷冻）或冷冻解冻的睾丸组织片段皮下移植到每只去势雄性 NCr 裸鼠的后腹侧，并在 3 个月后收获。从移植部位回收的新鲜新生睾丸移植物具有最先进的生精率，在 46.6%的曲细精管中具有伸长的精子和精子，并具有比所有其他冷冻解冻移植物组更高水平的血清睾酮（p<0.05）。与 PrOH、EG 和甘油相比，DMSO 组的新鲜移植物和冷冻解冻移植物在收获后具有最高的组织存活率（p>0.05）。基于组织病理学评估，与 EG 相比，DMSO 是冷冻和解冻新生小鼠睾丸的最有效 CPA（p<0.05）。与所有其他评估的冷冻保护剂相比，DMSO CPA 组获得的血清睾酮水平最高（p<0.05）。冷冻解冻移植物中观察到的典型损伤包括间质基质破坏、细胞间连接破裂以及精原细胞从基膜上脱离。这些发现表明，当使用含有 DMSO 的 HBSS 培养基冷冻保存新生小鼠睾丸时，睾丸最有效地保存下来，并且 CPA 的类型是获得冷冻保存、解冻和移植新生小鼠睾丸后最先进的生精和类固醇生成阶段的重要因素。
